Developmental coordination of mitochondrial dynamics and membrane remodeling drives organelle morphogenesis

Developmental coordination of mitochondrial dynamics and membrane remodeling drives organelle morphogenesis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Developmental coordination of mitochondrial dynamics and membrane remodeling drives organelle morphogenesis H Aravind , Vivek Kumar , Manish Jaiswal doi: https://doi.org/10.1101/2025.06.09.658059 H Aravind 1 Tata Institute of Fundamental Research Hyderabad , Ranga Reddy Dist. Hyderabad, 500046, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: manish{at}tifrh.res.in Vivek Kumar 1 Tata Institute of Fundamental Research Hyderabad , Ranga Reddy Dist. Hyderabad, 500046, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Manish Jaiswal 1 Tata Institute of Fundamental Research Hyderabad , Ranga Reddy Dist. Hyderabad, 500046, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: manish{at}tifrh.res.in Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Cellular morphogenesis involves the dynamic remodeling of mitochondria. For example, during spermatogenesis, mitochondria undergo distinct morphological changes as the cells transition from stem cells to mature sperms. However, how distinct processes regulating mitochondrial dynamics are developmentally coordinated to sculpt organelle morphology remains unclear. Using expansion microscopy to study Drosophila spermatogenesis, we first document a transition of tubular mitochondria into multilayered sheets organized in a sphere known as nebenkern, which then divides and remodels into two very long mitochondrial derivatives. We then show that the transition to organized multilayered structures is marked by membrane tubulation in the middle forming a division plane, the assembly of OPA1 and DRP1 rings along the division plane and a temporally synchronized downregulation of the fusion protein Mitofusin/Marf. We further show that Marf downregulation is mediated by the PINK1-PARK pathway, revealing an active suppression of fusion of mitochondrial derivatives during their segregation. Our study elucidates a developmentally programmed mechanism coupling membrane tubulation, fission, fusion, and membrane remodeling to drive mitochondrial morphogenesis during differentiation. Introduction Cellular morphogenesis is often coupled with extensive remodeling of mitochondria. Mitochondria undergoing dynamic changes — including fission, fusion, biogenesis, and selective clearance — that are tightly integrated with developmental processes across diverse systems ( 1 – 4 ). Transition of mitochondrial shape and size are important for meeting cellular metabolic demands but they can also affect signaling pathways and cell differentiation ( 5 , 6 ). However, despite its importance in cellular morphogenesis, the regulation of mitochondrial remodeling events that are developmentally programmed to achieve specialized size and shape has not been well characterized. Defective mitochondrial dynamics has been shown to cause developmental defects across various developmental contexts and models. Indeed, regulators of mitochondrial dynamics are known to contribute to lineage-specific differentiation programs. For example, Mitofusin-mediated mitochondrial fusion promotes cardiomyocyte differentiation through Notch activation ( 7 ), while OPA1, the Inner Mitochondrial Membrane (IMM) fusion protein, is required for FOXG1- dependent development of GABAergic neurons ( 8 ). Conversely, programmed mitochondrial fission via Drp1 regulates follicle cell differentiation in Drosophila by modulating Notch signaling and cell cycle exit ( 9 ). PINK1/Parkin-mediated mitophagy axis is also known to play critical roles in culling mitochondrial populations during tissue morphogenesis, e.g., midgut morphogenesis in flies ( 10 ). These studies highlight isolated aspects of mitochondrial dynamics involved in developmental regulation of mitochondrial morphology. However, how distinct processes such as fission, fusion, and membrane dynamics are temporally and spatially coordinated during development remains poorly understood. Mitochondrial remodeling is a critical aspect of spermatogenesis across diverse species. For instance, in mammals, mitochondria undergo a series of morphological changes, including elongation and condensation, ultimately forming the mitochondrial sheath that wraps around the flagellum’s midpiece, providing energy for motility ( 11 ). Similarly, in Drosophila , during early spermatid maturation, all mitochondria fuse into a giant spherical structure termed the nebenkern, which subsequently forms two mitochondrial derivatives that grow to a length spanning the sperm tail ( 12 ). Such remodeling of shape and size demands the precise orchestration of mitochondrial fusion, fission, and perhaps extensive membrane reorganization. Mutations in genes regulating mitochondrial fusion ( fzo, OPA1 ) or fission ( Drp1 ) regulators disrupt nebenkern morphology and impair spermatogenesis, underscoring the importance of mitochondrial remodeling during spermatogenesis ( 13 – 16 ). Here, we use high-resolution imaging approaches, including expansion microscopy, to capture the sequential stages of mitochondrial remodeling during Drosophila spermiogenesis. We uncover a developmentally programmed transition that synchronizes the downregulation of the fusion protein Marf (Mitofusin homolog) via the PINK1/Parkin pathway and the formation of concentric fission rings at the mitochondrial division plane that ensures precise remodeling of mitochondrial shape required for sperm morphogenesis. Results Expansion microscopy reveals structural details of mitochondrial remodeling during spermatogenesis To capture sequential transitions of mitochondria during spermatogenesis in flies in high resolution, we used expansion microscopy. To visualize the mitochondria, we used a genomically tagged Tom20::mCherry line, which marks the outer mitochondrial membrane. In the pre meiotic stages of spermatogenesis, mitochondria show filamentous morphology and are distributed throughout the spermatocytes ( Fig 1A (i)) . Expansion microscopy revealed that mitochondria in this stage have a short tubule-like morphology with occasional branching ( Fig 1B-B ’) . As these spermatocytes undergo meiotic division, mitochondria start aligning along the midplane of the two emerging daughter cells. During the telophase stage, a “bow-tie” shaped mitochondrial structure emerges ( Fig 1A (ii) ). Expansion microscopy of this arrangement revealed long mitochondrial tubules stacked along their length, forming a double nap cone structure. Their length spans both the daughter cells equally, with a constriction in the middle at the cytokinetic furrow giving it a characteristic “bow-tie” appearance ( Fig 1C-C ’ ). At the end of the second meiotic division, the mitochondrial tubules aggregate near one end of the nucleus, marking the beginning of the post-meiotic transitions. Download figure Open in new tab Fig 1: Expansion microscopy reveals finer morphological features of post-meiotic mitochondrial dynamics (A) Confocal image of various developmental stages and its associated mitochondrial morphology. (i) Meiotic stage, (ii) Meiotic division, (iii) Nebenkern, (iv) Elongating spermatid, (v) Mid-elongating spermatid, and (vi) elongated spermatids. Tom20::mCherry is used to stain the mitochondria. (B) Mitochondria in meiotic spermatids visualized with expansion microscopy. (B’) Volumetric rendering of a meiotic mitochondria. Arrows indicate branch points. This stage corresponds to region (i) in (A). (C) Bow-tie shaped mitochondria in the dividing meiotic spermatids visualized with expansion microscopy. (C’) Volumetric rendering of bow-tie mitochondria. Arrows indicate constriction along the cytokinetic furrow. This stage corresponds to region (ii) in (A). (D) “Early nebenkerns” with mitochondria in a spongiform arrangement visualized with expansion microscopy. (D’) Volumetric rendering of early nebenkerns clipped along x- and z- planes displaying a sphere of highly unordered sheet-like mitochondria. This stage corresponds to region (iii) in (A). (E) “Late nebenkerns” showing ordered whorls of mitochondria with constrictions on either ends visualized with expansion microscopy. (E’) Volumetric rendering of late nebenkerns clipped along x- and z- planes displaying several ordered sheets of mitochondria constricted along a plane. Scale bar represents 5μm in unless mentioned otherwise. A well-known feature of Drosophila spermatogenesis is the formation of a ball-like mitochondrial structure called the nebenkern ( Fig 1A (iii) ). While a typical confocal image of the nebenkern shows a round ball-like appearance, expansion microscopic images resolve nebenkerns to consist of two different morphologies ( Fig 1D,E ) . One type of nebenkern, as evident in 3-D rendering followed by clipping along the z- and x- planes, revealed an unordered arrangement of sheet-like mitochondria forming a sphere, exhibiting a spongiform appearance ( Fig 1D,D ’ ). The second type of morphology was consistent with the conventional images of nebenkern, wherein ∼7-8 whorls of mitochondrial sheets are arranged as concentric shells, known as the onion stage ( Fig 1E,E ’ ). 3-D rendering of late nebenkerns, but not early nebenkern, displayed a clear demarcation along the middle, running throughout its surface, bisecting it into two distinct hemispheres ( Fig 3A-F , Suppl. Video 1 ). We clipped this volume along the z- and x- direction and observed its morphology ( Fig 3B-F ) . The clippings showed that each whorl of the nebenkern is formed by mitochondrial membranes arranged as sheets. These sheets display constrictions and inter-whorl connections restricted only to the middle groove region, with very minimal inter-whorl connections observed in regions away from the middle groove. In these renderings, each sheet represents two sets of OMM and IMM separated by a narrow cytoplasm, as suggested by other published electron micrographs of the nebenkerns and our results ( Fig 3O-P ) ( 17 ) These transition events suggest that regulated mitochondrial dynamics lead to a transition of tubular mitochondrial forms to sheet-like forms, which exist in unordered spongiform arrangements in the early stages, followed by highly ordered whorls in the onion stage, which is prepared for subsequent division by forming a constriction in the middle groove. A programmed reduction of selective outer mitochondrial membrane proteins accompanies nebenkern maturation Germ cell development occurs within a cyst formed by two somatic cyst cells that ensheath them. We observed that mitochondrial morphology changes in a synchronized manner across all the germ cells within a cyst. This observation prompted us to reason that developmentally synchronized changes in the regulators of mitochondrial homeostasis might be driving these stage-specific changes. To study how these fine-tuned changes occur, we systematically investigated the dynamics of proteins involved in mitochondrial fission and fusion (Marf, OPA1, and Drp1), biogenesis (Tfam, Mdi, Larp, mtSSB, Tamas), activity (Complex V), protein import (Tom20), piRNA biogenesis (Daed and Gasz), and quality control (Pink1, Hsp60). Notably, we observed two distinct stages of round spermatids, characterized by nebenkerns displaying contrasting levels of four proteins, Marf::GFP ( Fig 2A-A ’’) , Mdi::GFP ( Fig S2A-A ’’) , Daed::GFP ( Fig S2B-B ’’) , and Gasz::GFP ( Fig S2C-C ’’) . While one population had high levels of these proteins ( Fig 2A,C white dashed lines) , the other population showed a marked reduction in the protein levels ( Fig 2A,C yellow dashed lines) . Further, their levels remained low in the elongated mitochondria, marking later stages of spermiogenesis ( Fig 2C orange dotted line) . To rule out the influence of the GFP tag, which may influence the localization and stability of proteins, we checked Marf levels in the nebenkerns of flies expressing a genomically tagged Marf::mCherry. Like Marf::GFP, Marf::mCherry expression showed two distinct populations of nebenkern mitochondria marked by high and low mCherry fluorescence ( Fig S3A-A ’’) . Further, immunostaining for HA epitope in flies expressing Marf::HA genomic rescue construct also showed similar results ( Fig S3B-B ’’) . Finally, we raised an antibody against Marf and performed immunostaining in wild-type CantonS flies. Immunostaining of Marf also showed two populations of nebenkern ( Fig S3C-C ’’) . Moreover, Marf expression, as measured by immunostaining with Marf antibody, correlated tightly with fluorescence levels of Marf::mCherry, indicating the specificity of the antibody ( Fig S3D-D ’’) . Together, our data suggest that a programmed reduction in Marf levels occurs during nebenkern development. Download figure Open in new tab Suppl. Fig S1: (Associated with Fig 1 ) (A-B) Multi-whorl organization of a late nebenkern imaged in SoRA-spinning disk super-resolution microscope without expansion. (B) Multi-whorl organization of a late nebenkern imaged after expansion. (C) Expansion factor was calculated by comparing the length of the major axis in late nebenkerns with or without expansion. n=15 nebenkerns from 3 independent experiments (non-expanded), n=17 nebenkerns from 3 independent experiments (expanded). p-value<0.0001. Statistical significance was calculated unpaired two-tailed t-test with Welch’s correction. Download figure Open in new tab Suppl. Fig S2: (Associated with Fig 2 ) (A) Two adjacent round spermatid cysts displaying high and low levels of Mdi in their nebenkern mitochondria. Density plot of Mdi intensity normalized to Complex V intensity (A’’). n=307 nebenkerns from 3 testis. Nebenkerns show Mdi::GFP (green) and Complex V (magenta). (B) Two adjacent round spermatid cysts displaying high and low levels of Daed in their nebenkern mitochondria. Density plot of Daed intensity normalized to Complex V intensity (B’’). n=230 nebenkerns from 7 testis. Nebenkerns show Daed::GFP (green) and Complex V (magenta). (C) Two adjacent round spermatid cysts displaying high and low levels of Gasz in their nebenkern mitochondria. Density plot of Gasz intensity normalized to Complex V intensity (C’’). n=168 nebenkerns from 4 testis. Nebenkerns show Gasz::GFP (green) and Complex V (magenta). (D) Two adjacent round spermatid cysts displaying high and low levels of Marf and Daed in their nebenkern mitochondria. Density plot of Marf intensity normalized to Daed intensity (C’’). n=301 nebenkerns from 5 testis. Nebenkerns show Daed::GFP (green) and Marf stained with Marf antibody (magenta). p- values<0.05 indicate a significant deviation from unimodality. Unimodality is estimated by Hartigans’ dip test. The scale bars indicate 5μm unless mentioned otherwise. Download figure Open in new tab Suppl. Fig S3: (Associated with Fig 2 ) (A-C’’) Two adjacent cysts displaying high and low levels of Marf (green) visualized with Marf::mCherry (A, A’’), Marf::HA (B,B’’), and Marf antibody (C-C’’). Complex V (magenta) is used to stain mitochondria (A’-A’’, B’-B’’, C’-C’’). (D-D’’) Marf stained with Marf antibody (green) and Marf::mCherry (red). Fluorescent signals from antibody and mCherry fluorescence shows good correlation as evident by their faithful replication of high and low levels of Marf in the two adjacent cysts. The scale bars indicate 5μm unless mentioned otherwise. Download figure Open in new tab Fig 2: Mitochondrial dynamics regulators undergo coordinated transitions during nebenkern maturation (A-A’’) Two adjacent round spermatid cysts displaying high and low levels of Marf in their nebenkern mitochondria. Density plot of Marf intensity normalized to Complex V intensity (A’’). n=184 nebenkerns from 6 testis. P-value suggests a significant deviation from unimodality. Nebenkerns shows Marf::GFP (green) and Complex V (magenta). (B-B’’) Two adjacent round spermatid cysts displaying high and low levels of Marf with uniform Tom20 levels in their nebenkern mitochondria. Density plot of Marf intensity normalized to Tom20 intensity (B’’). n=284 nebenkerns from 5 testis. P-value suggests a significant deviation from unimodality. Nebenkerns shows Marf::GFP (green) and Tom20::mCherry (magenta). (C-C’’) Two adjacent round spermatid cysts displaying high and low levels of Marf and Mdi in their nebenkern mitochondria. Density plot of Marf intensity normalized to Mdi intensity (C’’). n=139 nebenkerns from 3 testis. P-value suggests a non-significant deviation from unimodality. Nebenkerns show Mdi::GFP (green) and Marf::mCherry (magenta). (D-D’’) Marf levels in early nebenkerns with unordered sheet morphology. (E-E’’) Marf levels in late nebenkerns with ordered sheet morphology. Marf::GFP (green) and Tom20::mCherry (magenta) imaged using SoRa super-resolution microscope with deconvolution. (F-F’’) Two populations of OPA1 showing dispersed and ring morphology. Proportions of nebenkerns with dispersed or ring-like localization of OPA1 is quantified in (F’’). n=371 nebenkerns from 15 testis. Chi-Squared analysis with Yates’ correction shows a significant association of OPA1 localization change to increase in aspect ratio of nebenkerns (p-value: <0.0001). (G-H’’) OPA1 rings in two different orientations. OPA1::HA in green, ComplexV in magenta. (I-I’’) OPA1 localization in high and low Marf containing nebenkerns. (F’’’) Quantification of Mean marf intensity in OPA1 populations showing distinct localization patterns. n=237 nebenkerns from 6 testis. Error bar represents Standard Error of Mean. Unpaired two tailed t-test with Welch’s correction used for calculating significance. Marf::GFP in magenta, OPA1::HA in green (J-J’’) Two populations of DRP1 showing dispersed and ring morphology. Proportions of nebenkerns with dispersed or ring-like localization of DRP1 is quantified in (J’’). n=372 nebenkerns from 14 testis. Chi-Squared analysis with Yates’ correction shows a significant association of DRP1 localization change to increase in aspect ratio of nebenkerns (p-value: <0.0001). (K-L’’) DRP1 rings in two different orientations. DRP1::HA in green, ComplexV in magenta. (M-M’’) DRP1 localization in high and low Marf containing nebenkerns. (M’’’) Quantification of Mean marf intensity in DRP1 populations showing distinct localization patterns. n=256 nebenkerns from 4 testis. Error bar represents Standard Error of Mean. Unpaired two tailed t-test with Welch’s correction used for calculating significance. Marf::GFP in magenta, DRP1::HA in green. Multimodal distributions in (A’’) and (B’’) suggest multiple populations with varying Marf intensity. Unimodal distribution in (C’’) suggests Marf and Mdi levels are uniformly downregulated. Unimodality is estimated by Hartigan’s dip test. Scale bar represents 5μm. All four proteins we identified to undergo reduction during nebenkern development have different mitochondrial functions; however, they are all OMM proteins. Therefore, we asked if the downregulation in Marf is temporally correlated with the downregulation of other proteins. To check this, we immunostained for Marf in testis expressing Mdi::GFP ( Fig 2C-C ’’) or Daed::GFP ( Fig S2D-D ’’) . The reduction in Marf levels showed a high correlation with the reduction of other OMM proteins as the nebenkern transitions to elongation. Finally, we asked if all OMM proteins follow a similar trend. To check this, we monitored the levels of another OMM protein - TOM20 using flies expressing genomically tagged Tom20::mCherry. We find that Tom20::mCherry levels remained consistently high in the early and late nebenkern stage, marked by changes in Marf levels ( Fig 2B-B ’’) . Together, our results suggest a selective reduction in the levels of some mitochondrial outer membrane proteins as the nebenkern transitions to elongate. Based on these observations, we suggest that round spermatids can be classified into two types marked by a transition in the nebenkern protein levels. During this transition, “early” nebenkerns undergo a coordinated reduction in at least four mitochondrial proteins to form the “late” nebenkern stage before elongation. We term this transition as nebenkern maturation. Mitofusin/Marf levels are developmentally reduced during nebenkern maturation Next, we asked if Marf downregulation is associated with the transition of nebenkerns from the early unordered sheet stage to the late ordered whorl stage which is followed by its division. To check this, we expressed Tom20::mCherry in testis expressing Marf::GFP and performed SoRa super-resolution imaging followed by deconvolution. This approach allowed us to visualize nebenkern whorls without the need for expansion. We observed that early nebenkerns with unordered sheet morphology displayed high Marf levels ( Fig 2D-D ’’) . However, the late nebenkerns with ordered sheet morphology displayed very low Marf levels ( Fig 2E-E ’’) . Together, these results indicate that Marf downregulation is associated with the morphological stage transition of nebenkerns during their maturation. OPA1 and DRP1 organized in a ring-like structure around nebenkern Since Marf is downregulated, we expected the IMM fusion protein OPA1 to show a similar pattern in order to coordinate mitochondrial dynamics during nebenkern maturation. However, immunostaining of OPA1::HA revealed two distinct OPA1 localization patterns in the nebenkerns. While one population of these nebenkerns shows a uniform expression of OPA1 throughout their surface, the other population shows OPA1 localized as a ring-like structure around the nebenkern ( Fig 2F-H , suppl video 2) . As the nebenkerns started to elongate, marked by increasing aspect ratio, the proportion of nebenkerns with OPA1-ring steadily increased ( Fig 2F ’’) . Interestingly, the OMM fission protein DRP1 displayed localization patterns closely resembling OPA1. Immunostaining of genomically tagged DRP1::HA revealed that one population of round spermatids shows DRP1 assembled as punctate structures decorating the entire nebenkern surface. The second population, however, showed a distinct ring-like structure circumferencing the nebenkern ( Fig 2J-L ’, suppl. Video 3) . Similar to OPA1 rings, elongating populations of nebenkerns were increasingly associated with DRP1 rings, suggesting DRP1-ring formation is a feature of nebenkern elongation ( Fig 2J ’’) . Since OPA1/DRP1-ring formation is associated with only half of the nebenkern population, we asked if it coincided with Marf downregulation and, hence, with nebenkern maturation. Co-staining of Opa1::HA or DRP1::HA in Marf::GFP expressing nebenkerns showed that nebenkerns with high Marf levels exhibited a uniform distribution of OPA1/DRP1 throughout their surface ( Fig 2I-I ’’, white dashed line). However, the proportion of nebenkerns with OPA1/DRP1 rings increased as Marf levels declined ( Fig 2I ’’) . This suggests that OPA1/DRP1 ring formation is associated with nebenkern maturation. Moreover, these rings may form along nebenkern constriction sites, bisecting them as they begin to elongate and divide. Structural transitions of nebenkern are marked by organised inter-whorl connections and central constriction zones Previous studies suggest that nebenkern comprises two large mitochondria wrapped around each other, which subsequently unfurl into two derivatives ( 12 , 17 ). Our observations, however, indicate that an active division may divide the nebenkern at the middle groove region. To resolve this, we studied the membrane organizations in the late nebenkern stage using expansion microscopy. Strikingly, we observed that constriction points in a given whorl are associated with the regions where adjacent whorls connect with each other or with farther whorls ( Fig 3K-N , Suppl. Video 4) . To understand the relationship between these two features, we performed volumetric rendering of an isolated inter-whorl connection away from the groove region ( Fig 2G-J , Suppl. Video 5) . We observe that when two whorls are connected, they traverse a whorl in the middle ( Fig 3G,I ) . This traversion results in a tubular, bridge-like structure separating the lumen of the middle whorl ( Fig 3I ) . When clipping one of the whorls connected by the bridge, it is evident that the bridge forms a curved region on the inner surface of the sheets constituting the middle whorl ( Fig 3H ) . Meanwhile, the outer surface of the same sheet displays a hole at the site of bridge formation ( Fig 3G ) . Consequently, we consider that holes on the surface of a given sheet indicate a connection made by the whorl consisting of this sheet to other whorls. Meanwhile, a region of positive curvature on the surface of the sheet indicates an inter-whorl connection traversing through the whorl consisting of this sheet. Download figure Open in new tab Fig 3: Alternative fusion of whorls along middle groove tubulate sheet-like mitochondria in nebenkern (A-F) Volumetric rendering of a late nebenkern. The volumes are unclipped (A) or clipped at different depths along the z-plane (B and C), and are rotated 50 0 along the y-axis and clipped along the x-plane (D-F). Constrictions of all sheets are restricted to the middle groove region (marked with orange dashed lines). (G-J) Volumetric rendering of an inter-whorl connection traversing a middle whorl. The connection resembles a hole from the top view (G) and a tube from the side (I) (rotated 100° along the z-axis). (H) Clipping the top-sheet in (G) shows a region of positive curvature in the lower sheet. (J) Clipping (I) along the x-plane shows the inter-whorl connection constricting the lumen of the middle whorl. (K-N) Four adjacent shells, numbered 1 to 4 together, make up three whorls marked i to iii. A multi-shell branch point, formed by the mixing of several shells, is marked by a dotted line. Each inter-whorl connection made is marked with a green plus. Each separation is marked with white star. White arrow indicates a hole on shell 4 formed through an inter-whorl connection. (O-P) 2-D Schematic of inter whorl connections tubulating the sheets. (P) shows the cytoplasmic bridges between two sets of OMM and IMM making up each whorl. C.B- cytoplasmic bridges. M- matrix. Scale bar represents 5μm. When we examined the middle groove region closely, we could readily appreciate that each whorl made connections with several others, each time traversing through its adjacent whorl ( Fig 3K-N , Suppl. Video 4) . Moreover, these connections resulted in a hole-like structure on the sheets of the whorls they traversed, confirming the membrane features observed in our model of the isolated interconnection ( Fig 3N , arrow) . We reason that such an arrangement would help achieve two aspects in terms of dividing the nebenkern. First, it would assist in tubulating the sheet-like whorls, which can readily be divided. We hypothesize this, as the currently known mitochondrial division machinery, including DRP1, stabilizes on tubular structures ( 18 ). Second, multiple traversals along a plane in a sheet would result in the constriction of the sheet along that plane. If the adjacent tubules were to close, it would lead to the separation of the lumen of the whorls into two distinct regions. OPA1 and DRP1 define a mitochondrial fission ring along the middle groove The expansion microscopy data indicates the presence of tubular bridges traversing each whorl of the nebenkern in the middle groove region ( Fig 3G-J ) . The presence of DRP1 and OPA1 along the middle groove region prompted us to hypothesize that DRP1 might help in dividing the tubular bridges while OPA1 might help in fusing the bridges together within the whorls, thereby enabling the separation of the nebenkern into two mitochondrial derivatives. Thus, we decided to investigate whether OPA1 and DRP1 rings are indeed formed at nebenkern constriction sites. We immunostained for either OPA1::HA or DRP1::HA in nebenkerns marked with Tom20::mCherry and performed SoRa super-resolution microscopy. We observed that the dispersed localization of OPA1 and DRP1 is associated with early nebenkerns with unordered sheet morphology ( Fig 4A-A ’’, C-C’’) . However, as the ordered sheets form in the late stages, OPA1 and DRP1 are intensely localized along the middle groove region ( Fig 4B-B ’’, D-D’’). This suggests that OPA1 and DRP1 localization dynamics are associated with morphological transitions during nebenkern maturation. Download figure Open in new tab Fig 4: OPA1 and DRP1 forms fission ring along constriction sites in the middle groove of late nebenkerns (A-A’’) Dispersed localization of OPA1 in early nebenkerns. (B-B’’) Enrichment of OPA1 in the constriction sites in late nebenkern. (E-E’) OPA1 localization in the apical surface of a late nebenkern. (F-F’) OPA1 localization in the medial plane of the same late nebenkern. White box indicates the inset. Arrows indicate constricted regions. OPA1::HA (green) and Tom20::mCherry (magenta). (C-C’’) Dispersed localization of DRP1 in early nebenkerns. (D-D’’) Enrichment of DRP1 in the constriction sites in late nebenkern. (G-G’) DRP1 localization in the apical surface of a late nebenkern. (F-F’) OPA1 localization in the medial plane of the same late nebenkern. White box indicates the inset. Arrows indicate constricted regions. DRP1::HA (green) and Tom20::mCherry (magenta). The scale bars indicate 5μm unless mentioned otherwise. In the late nebenkerns, distinct OPA1 punctae were present across several adjacent whorls of mitochondria undergoing constriction, giving the appearance of a complete ring ( Fig 4E-F ’) . On the apical surface of the nebenkern, OPA1 is intensely localized to nebenkern constriction sites that appear like short tubules ( Fig 4E , zoomed insets) . In the medial plane, OPA1 is preferentially localized along the nebenkern division plane, along the constricted regions of each whorl ( Fig 4F , zoomed insets) . This suggests that OPA1 accumulates at nebenkern constriction sites within each whorl. DRP1 localized intensely along the nebenkern constriction sites, similar to OPA1 ( Fig 4G-H ’) . On the apical surface, DRP1 punctae localized next to narrow tubules ( Fig 4G , zoomed insets) . In the medial plane, DRP1 localized only to nebenkern constriction sites, mainly along the middle groove ( Fig 4H , zoomed insets) . Individual DRP1 puncta could be observed across each whorl of nebenkern simultaneously. This indicates that each whorl undergoes division individually; however, the position of these divisions is uniform. Together, we propose that nebenkern formation involves the aggregation of several tubular mitochondria, which undergo regulated fusion events to form sheets in a spongiform arrangement ( Fig 1D,D ’) that further fuse and intertwine to create the well-recognized multi-whorl organization ( Fig 1E,E ’) . Once formed, they exhibit constriction and inter-whorl connections through the middle of the nebenkern, resulting in two distinct hemispheres (Suppl. Video 4,5) . Since the inter-whorl connections are restricted to the middle groove region, we propose that it induces regions of discontinuity along an approximately central plane within whorls, possibly aiding in their division. Overall, the tubulated inter-whorl connections and middle-groove constrictions may define the division site. Finally, OPA1 and DRP1 rings are localized to nebenkern constriction sites, suggesting an active division of nebenkerns. We term the DRP1 ring around matured nebenkerns as fission-ring. PINK1 mediates Marf downregulation prior to mitochondrial fission As shown in Figure 2 , dramatic downregulation of Marf coincides with the formation of DRP1/OPA1 fission-ring. Marf downregulation, along with Drp1 and Opa1 translocation, could be crucial for the successive fission of the nebenkern. Previously, PINK1, which is shown to regulate mitochondrial dynamics and quality control, was found to be required for nebenkern division. Stabilization of PINK1 on the OMM is known to activate mitophagy, induce Marf degradation independent of mitophagy [19,20], and promote mitochondrial fission through DRP1 phosphorylation [21,22]. In our investigation of the localization of mitochondrial dynamics proteins, we observed that PINK1 levels increase in nebenkern and remain constant during nebenkern maturation ( Table 1 and Supp Fig S4A-A ’) , starkly contrasting with other OMM proteins undergoing downregulation. Hence, we asked if PINK1 upregulation mediates Marf downregulation and/or OPA1/DRP1 ring formation in maturing nebenkerns. Download figure Open in new tab Suppl. Fig S4: (Associated with Fig 5 ) (A) PINK1 levels in spermatocytes, nebenkerns, and elongating mitochondrial derivatives. PINK1::9Myc is visualized with Myc staining (magenta) and counterstained with DAPI (cyan). (A’) Comparative increase in PINK1 levels during the nebenkern and elongating stages normalized to spermatocytes and visualized with Fire LUT. (B-C’) Marf levels in control ( park[21],Marf::HA/+) and park (park21,Marf::HA/park[13]) mutants. Density profile of Marf normalized to ComplexV in control (B’’) and park mutants (C’’). n=263 nebenkerns from 5 testis in control (B”) and n=367 nebenkerns from 6 testis in mutant (C’’). p-value<0.05 indicates a significant deviation from unimodality. Unimodality is estimated by Hartigans’ dip test. Marf in green. ComplexV in magenta. Arrows indicate elongating spermatids. (D-D’’) Marf and Gasz levels in PINK1 mutants. Gasz levels (D) are downregulated in the nebenkerns of two adjacent cysts while Marf levels (D’) are unchanged. Nebenkerns show Gasz::GFP (green) and Marf (magenta). Density profiles of Gasz normalized to Complex V show multimodal distribution (E) while Marf normalized to ComplexV shows a unimodal distribution (F). n=322 nebenkerns from 4 testis. Unimodality is estimated by Hartigans’ dip test. The scale bars indicate 5μm unless mentioned otherwise. Suppl. Fig S5: (Associated with Fig 5 ) (A-A’) Early elongating spermatids in control shows two separated derivatives filled with MDiVs. Expansion microscopy image in (A) along with volumetric rendering in (A’). (B-B’) Early elongating spermatids in PINK1 mutants shows a single derivative filled with MDiVs. Expansion microscopy image in (A) along with volumetric rendering in (A’). The scale bars indicate 5μm. View this table: View inline View popup Table 1: Genotypes used in the study First, we asked whether OPA1/DRP1 ring formation is affected in Pink1 mutants. Immunostaining of OPA1::HA or DRP1::HA in Pink1 mutant testis revealed OPA1/DRP1 rings around a subset of nebenkerns ( Fig 5A-B ’’) . Moreover, the proportion of OPA1/DRP1 rings increased with the aspect ratio, suggesting that PINK1 is neither required for fission-ring assembly nor for initiating division of the maturing nebenkern ( Fig 5A ’’,B’’) . Download figure Open in new tab Fig 5: PINK1 mediated Marf downregulation during nebenkern maturation prevents unopposed fusion of mitochondrial derivatives (A-A’) OPA1 localization in early and late nebenkerns. (A’’) Quantification of OPA1 localization during nebenkern elongation. n=294 nebenkerns from 11 testis. Chi-Squared analysis with Yates’ correction shows a significant association of OPA1 localization change to increase in aspect ratio of nebenkerns (p-value: <0.0001). OPA1::HA in green. ComplexV in magenta. (B-B’) DRP1 localization in early and late nebenkerns. (B’’) Quantification of DRP1 localization during nebenkern elongation. n=233 nebenkerns from 8 testis. Chi-Squared analysis with Yates’ correction shows a significant association of DRP1 localization change to increase in aspect ratio of nebenkerns (p-value: <0.0001). DRP1::HA in green. ComplexV in magenta. (C-D’) Marf levels in control and pink1 mutants. Density profile of Marf normalized to ComplexV in control (C’’) and pink1 mutants (D’’). n=208 nebenkerns from 6 testis in control (C’’) and n=197 nebenkerns from 5 testis. Unimodality is estimated by Hartigans’ dip test. P-value <0.05 suggests a significant deviation from unimodality. Marf in green. ComplexV in magenta. Arrows indicate elongating spermatids. (E-H) Volumetric renderings of early and late nebenkern morphology determined by expansion microscopy and clipped along z- and x-axis in control (E-early, G- late) and in pink1 mutants (F-early, H-late). Mitochondrial membranes marked by Tom20::mCherry. (I-J’) Volumetric renderings of early elongating nebenkern morphology determined by expansion microscopy and clipped along the z-axis in control (I-I’) and in pink1 mutants (J-J’). White box indicates insets. Control (I’) nebenkern shows distinct outer shells while pink1 mutant (J’) shows fused outer shells. Mitochondrial membranes marked by Tom20::mCherry. (K) Walkthrough along z-direction of the surface of a pink1 mutant nebenkern showing looping and re-fusion of the outer shells in progress. Mitochondrial membranes marked by Tom20::mCherry. The scale bars indicate 5μm unless mentioned otherwise. Next, we asked if Marf downregulation in maturing nebenkerns requires PINK1. Marf immunostaining revealed that Pink1 mutants did not show two nebenkern populations with contrasting levels of Marf, in contrast to the wild-type nebenkerns ( Fig 5 C-D’) . Density plots of normalized Marf intensity showed a unimodal distribution in Pink1 mutants compared to a bimodal distribution in controls ( Fig 5C ’’,D’’) . This suggests that Marf downregulation is absent in Pink1 mutant nebenkerns. Moreover, Marf levels remained consistently high in early elongating nebenkerns of Pink1 mutants ( Fig 5D ’, arrow) . These results suggest that PINK1 is required for Marf downregulation in maturing nebenkerns. Parkin, an E3 ubiquitin ligase, acts downstream of PINK1 to regulate mitochondrial dynamics ( 19 , 20 ). PINK1 is known to recruit Parkin to the OMM, which in turn ubiquitinates OMM proteins and induces their degradation [19–21]. Therefore, we hypothesized that Marf downregulation in maturing nebenkerns might be absent in Park mutants. Akin to Pink1 mutants, Park mutants displayed high levels of Marf in nebenkerns ( Fig S4 B-C’’) . Moreover, Marf levels were visibly upregulated in the early elongating stages ( Fig S4C-C ’, yellow dotted line) . Thus, we propose that the developmental activation of the PINK1/Parkin axis during nebenkern maturation mediates the specific downregulation of Marf, which may be crucial for enabling the segregation of mitochondrial derivatives in later stages. Since Marf downregulation during nebenkern maturation requires PINK1, we checked if PINK1 mediates the downregulation of other OMM proteins during nebenkern maturation. We found that in Pink1 mutants, Gasz::GFP intensity showed two distinct nebenkern populations, while Marf levels remained uniform in both populations ( Fig S4D-F ) . This result suggests that the downregulation of OMM proteins other than Marf occurs independently of PINK1. This also suggests that although the developmental events marking nebenkern maturation, including the downregulation of OMM proteins and fission-ring formation, are coordinated, they are independently regulated. PINK1 prevents refusion of the elongating mitochondrial derivatives Since Marf is not downregulated in PINK1 and Park mutants, we hypothesized that the nebenkern division defect observed in Pink1 mutants during spermiogenesis may be caused by refusion of the separating derivatives because of increased Marf levels. To determine if Pink1 mutant nebenkerns divide and re-fuse, we employed expansion microscopy and used Tom20::mCherry to mark the mitochondrial membrane. We observed no marked changes in the morphology of early and late nebenkerns in Pink1 mutants compared to controls ( Fig 5 E-H) . Volumetric renderings of expanded nebenkerns suggest that the early stages were marked with a spongiform arrangement of several short sheets in both control and Pink1 mutant nebenkerns ( Fig 5E-F ) . Similarly, the late nebenkerns displayed bifurcations along the middle groove marked by constrictions and inter-whorl connections in both control and Pink1 mutants ( Fig 5G-H ) . These results are consistent with the observation that OPA1 and DRP1 dynamics are unaffected in Pink1 mutants ( Fig 5A-B ’) . This suggests that PINK1 is not required for initial membrane reorganization and constriction along the middle groove. However, morphological defects in Pink1 mutant nebenkerns became apparent as they started showing early signs of elongation. Volumetric rendering followed by clipping along different z-planes suggests that the fusion of outer whorls in Pink1 mutants results in a continuous outer whorl despite the inner whorls being constricted ( Fig 5J-J ’) . Contrarily, in control, the outer whorls remain separated and get constricted, coordinating with the inner whorl constrictions ( Fig 5I-I ’) . We then resorted to volumetric rendering of expansion microscopic images to capture static images that might hint at refusion of outer whorls. We analyzed the surface of an early elongating Pink1 mutant nebenkern after clipping the volume along the z-direction. We observe tubular projections arising from the outer whorls on either side of the middle groove, contacting and merging, seemingly to form a bridge between the two outer whorls ( Fig 5K ) . We hypothesize that several such events might occur in Pink1 mutants at different positions along the middle groove due to increased fusion potential in these nebenkerns, thereby fusing the outer whorl together. Discussion In this study, we provide a high-resolution dissection of mitochondrial remodeling during spermiogenesis, revealing how giant post-meiotic mitochondria undergo a sequence of architectural transitions—from massive fusion into multilayered whorls, to division via a precise fission plane, and ultimately to elongation. Prior to elongation, the giant mitochondria (the nebenkern) must divide into two mitochondrial derivatives. This division presents two fundamental challenges: a stoichiometric challenge, due to elevated fusogenic potential from abundant outer mitochondrial membrane fusion proteins, Marf and Fzo, which are essential for nebenkern formation but may drive refusion during division; and a geometric challenge, as multiple concentric mitochondrial sheets must be simultaneously and uniformly separated. We show that this transition is developmentally programmed and is marked by spatially and temporally synchronized downregulation of Marf/Mitofusin via the PINK1/Parkin pathway, thereby reducing fusion potential. Concurrently, tubulation of mitochondrial sheets at the mid-plane appears to facilitate DRP1 and OPA1 recruitment, enabling localized membrane fission and fusion. This coordinated strategy appears to overcome both stoichiometric and geometric constraints, enabling the division and elongation of giant mitochondria in a developmentally regulated manner. Developmental synchronization of mitochondrial transitions It is intuitive to presume that mitochondrial dynamics regulators must work synchronously and respond to cell-intrinsic and extrinsic cues to modulate developmentally dictated mitochondrial transitions. However, models to study these coordinated transitions are not well defined. Here, we show temporally synchronized changes in mitochondrial fission (DRP1), fusion (Marf and OPA1), and quality control regulators (PINK1) to coordinate the formation and division of a complex mitochondrial assembly called the nebenkern during Drosophila spermatogenesis. It remains to be understood how these distinct arms of mitochondrial dynamics are regulated to temporal perfection during nebenkern maturation. We suspect that non-cell-autonomous signaling from surrounding somatic cyst cells could regulate the widespread changes in mitochondrial dynamics in the germ cells. We base our hypothesis on the following sets of data: ( 1 ) In PINK1 mutants, only Marf downregulation, but not the other events associated with nebenkern maturation, is affected, highlighting independent mechanisms regulating mitochondrial remodeling. ( 2 ) Nebenkern maturation, characterized by Marf downregulation and fission ring formation, proceeds synchronously in all 64 spermatids within a given cyst. These synchronous events thus hint at the possibility of external signaling regulating these processes, possibly from cyst cells. It will be interesting to identify these signals and how cyst cells may time the secretion of these signals. Unveiling how these factors induce synchronous changes in mitochondrial fission and fusion might help us understand the basal regulation of these fundamentally opposing processes in other cellular contexts. Role of PINK1 pathway beyond quality control The role of PINK1 in activating mitophagy in response to catastrophic mitochondrial insults, like CCCP-induced mitochondrial depolarization, is well established in vitro. In contrast, studies exploring the role of PINK1 in vivo hint at its roles beyond mitophagy, involving diverse mechanisms including regulating mitochondrial surface translation, inhibiting fusion, and activating fission ( 21 – 25 ). An emerging trend from these in vivo studies is that PINK1 can regulate mitochondrial dynamics independent of its role in quality control. Unlike mitochondria in other tissues, sperm mitochondria require two OMM fusion genes ( marf and fzo) for fusing the nebenkern. Fzo protein is observed only during the nebenkern stage and disappears during the subsequent elongation stages ( 14 ). This suggests that nebenkern mitochondria are highly “sticky” to begin with, as they aggregate and fuse. In the later stages, when mitochondria is ready to divide, we suggest that the stoichiometric challenge of heightened fusion is overcome by PINK-mediated Marf downregulation. This would ensure the nebenkerns are “less sticky” as they are divided and prevent their refusion during segregation. Our results suggest that PINK1 specifically limits the unopposed fusion of the dividing derivatives without affecting their fission. Recently, we discovered that PINK1/Parkin-induced degradation of Marf in lrpprc2 mutants is essential for segregating healthy and dysfunctional mitochondria, thereby influencing cellular health ( 23 ). Here we show that PINK1-mediated Marf degradation is required for segregating mitochondrial derivatives in developing sperms under physiological conditions. Together, we suggest that context-specific activation of PINK1 can specifically modulate Marf levels on mitochondria to regulate their fusion. We propose that, in addition to regulating mitochondrial quality control, PINK1 can directly regulate mitochondrial dynamics by decreasing Marf levels; a process that appears to be employed during development, mitochondrial stress, and disease. Forming and dividing a large mitochondria Earlier observations of the nebenkern using electron microscopy showed thin constrictions that established a bridge between the two hemispheres of the nebenkern ( 26 ). These bridges were thought to be evidence of two previously segregated mitochondria intertwined with each other, possibly because mechanisms of mitochondrial division were not established at the time. Consequently, it was proposed that nebenkerns unfurl as they separate into two mitochondrial derivatives. We propose to revise this model of nebenkern division by suggesting that nebenkerns undergo active division to give rise to two mitochondrial derivatives instead of unfurling as two intertwined mitochondrial derivatives. However, the nebenkern mitochondria exist as several layers of sheets. This imparts a geometric constraint as mitochondrial division requires the mitochondria to have tubular morphology. In fact, several studies have demonstrated that the constriction of tubular membranes is required for higher order Drp1 oligomerization forming a ring around the constricted membrane causing its fission ( 27 , 28 ). Our model of nebenkern division overcomes this geometric constraint by suggesting that thin bridges connecting two alternate whorls in the nebekern could help tubulating the sheet-like whorls, which can then readily be divided. The bridges also effectively constrict the lumen of the whorls through which they pass. Volumetric rendering of the entire nebenkerns clearly demonstrates that this process is localized along the middle groove. The presence of DRP1 along these sites also lends support to the idea that nebenkerns could be divided along the middle groove. Indeed, super-resolution imaging of dividing nebenkerns shows DRP1 punctae assembling across multiple whorls of nebenkern. The formation of OPA1-ring, which colocalized with DRP1, remains an intriguing yet exciting observation. Based on our expansion microscopy data, we hypothesize that accumulated OPA1 along the mid groove could help in the fusion of adjacent intra-whorl bridges, thereby separating the lumen of the sheets across the two hemispheres. Consequently, the coordinated dynamics of OPA1 and DRP1 would be necessary for the efficient separation of the nebenkern. Together, our findings reveal a mechanism that integrates fusion suppression, fission activation, and membrane remodeling to reshape mitochondria during cellular morphogenesis. Similar mechanisms may operate in other developmental or stress contexts where organelle architecture must adapt to cellular demands. Limitations of the study Although our results show a clear correlation between OPA1 and DRP1 localizations in the form of a ring along nebenkern constriction sites, it is still unclear if OPA1 accumulation is important for the induction of the constrictions and hence DRP1 recruitment. We were not able to test this idea as nebenkerns do not form completely in OPA1 mutants. The impact of Marf upregulation on spermiogenesis failure in a wild-type context is also unclear. Overexpressing Marf using a germ cell-specific GAL4 was not enough to phenocopy PINK1 mutants, as Marf was downregulated in the nebenkerns despite being overexpressed in the early stages. Materials and Methods Fly husbandry Flies were cultured on standard media containing sucrose, malt, yeast and corn flour at room temperature. Crosses were maintained at 25°C. The genotypes used in this study are provided in Table 1 . Expansion microscopy of Drosophila testis The expansion microscopy protocol was adapted from Asano SM et.al.(Asano et al., 2018). Briefly, immunostained testis were incubated in 0.1 mg/ml Acryloyl-X SE (AcX) (Invitrogen A20770) in PBS (prepared fresh from 10 mg/ml AcX in DMSO (Sigma 276855) at room temperature overnight. Following this, the tissues were washed in 1X PBS twice for 15 minutes each. After washing, the tissues were incubated in the gelling solution (Stock X ( Table 2 ), 0.5% 4HT (Sigma 176141), 10% TEMED (Himedia MB026), 10% APS (Himedia MB003), in 47:1:1:1 ratio) at 4°C for 30 minutes in the dark. For polymerization, the tissues were transferred to slides containing 200-300 μl of gelling solution and gently compressed using a parafilm-wrapped coverslip. The gelation was performed by incubating this setup at 37oC for two hours in a humidified chamber. After polymerization, the gel was cut into smaller pieces containing not more than two tissues per piece, and incubated in the digestion buffer ( Table 3 ) containing Proteinase K (Himedia MB086) (final concentration of 8U/ml) for digestion. The digested gel pieces were then stored in 1X PBS at 4°C prior to expansion. Expansion of the digested gel was performed just before imaging. For expansion, the gels were incubated in milliQ water in a large petri dish. Water changes were performed at an interval of 20 minutes thrice. The expanded gels were carefully transferred to a cover glass, and extra liquid was removed with tissue paper. To prevent the gel from drifting while imaging, 0.5% agarose solution (Sigma-Aldrich A9045) was added to the boundaries of the gel. View this table: View inline View popup Download powerpoint Table 2: Composition of Stock-X solution View this table: View inline View popup Download powerpoint Table 3: Composition of Digestion buffer Imaging of the expanded gels was performed using 60X water immersion objective with 4X magnification by Lens-switched light path in SORA (Spinning Disk Super Resolution by Optical Pixel Reassignment) mode using an inverted Nikon CSU-W1 SORA spinning disk confocal microscope. A few drops of milliQ were added frequently to moisturize the gel and prevent it from shrinking during the imaging. Immunostaining The testis were dissected in 1X PBS and fixed with 4% paraformaldehyde solution in 1X PBS (Himedia TCL119). Following this, the testis were washed with 0.2% PBST (Triton X-100 (Himedia MB031) in 1X PBS) and incubated with primary antibodies of indicated dilutions overnight at 4oC. This was followed by three washes in 0.2% PBST. The testes were incubated in blocking solution containing 5% Goat serum diluted in 0.2% PBST for 2 hours at room temperature. Following blocking, the testes were incubated in secondary antibodies of the indicated dilutions for 2 hours at room temperature. The testes were then washed with 0.2% PBST thrice at room temperature, ten minutes each. The testes were then mounted on a glass slide with a drop of VectaShield (Vector labs H-1900-10). All antibody dilutions were made in 0.2% PBST. A list of antibodies used in the study are provided in Table 4 . View this table: View inline View popup Download powerpoint Table 4: List of antibodies used in the study Image processing and analysis 3-D rendering Volumetric rendering of all images was performed using “3D script” plugin in ImageJ ( 29 ). To increase the smoothness of the rendered volumes along the z-direction, images were scaled along z-direction with factor 5.0 using Bilinear Interpolation without altering the aspect ratio. Background in the projected images were removed by marking the regions using the “free-hand” tool in ImageJ followed by “clear selection” dialog in 3D script. For rendering with multiple channels, “Combined transparency” function was used for visualization. Expansion factor calculation To calculate the expansion factor, we compared the increase in the major axis of nebenkerns with and without expansion. To ensure uniformity in the developmental stage used for this comparison, we measured the major axis of late nebenkerns. The quantified data are pooled from three independently performed expansion experiments. Deconvolution Images were deconvolved in NIS-Elements software (5.42.01 (Build 1793)) using the Richardson-Lucy algorithm. The number of iterations was determined automatically. Segmentation of nebenkerns Segmentation of nebenkerns was performed in ImageJ using a custom macro. The nebenkern-containing regions were marked with the “free-hand tool” in the Z-projected images (maximum projection). The region was then duplicated, and the unmarked areas were removed using the “Clear Outside” tool. We performed Gaussian Blur (sigma = 5) followed by Auto thresholding to create masks of the nebenkerns. The masks were filtered using the size parameter set to 10-Infinity to identify the nebenkerns. The masks were overlaid on the original z-projected images to measure Mean intensity and Aspect Ratio across all channels using the “multi-measure” tool. Identification of distinct nebenkern populations To determine if the observed normalized intensity values of nebenkerns were sampled from different populations, we plotted these values as a probability density function using R. The bandwidth was estimated using the Freedman-Diaconis rule. We then performed Hartigans’ dip test to determine if the curve was unimodal or multimodal. A multimodal curve would indicate the presence of distinct populations of nebenkerns. Statistical analysis In all experiments, n represents the number of nebenkerns. The number of testis used for each experiment is mentioned in the figure legends. Deviation from unimodality in all density plots was determined using Hartigan’s dip test in R. P-values below 0.05 indicate a significant deviation from unimodality. Categorical data were measured for association using the Chi-Squared test with Yates correction. P-values below 0.05 indicate significant association. All other data were analyzed using two-tailed unpaired t -tests with Welch’ s correction. Chi-Squared analysis and t -tests were performed using GraphPad Prism 10. Author Contribution A.H. and M.J. conceived the study. A.H. designed and performed all experiments, analyzed data, and wrote the manuscript. A.H. and V.K. conducted the expansion microscopy experiments. M.J. supervised the project, secured funding, provided overall guidance, and contributed to manuscript writing and revision. All authors discussed the results and approved the final version of the manuscript. Acknowledgements We thank Hong Xu, Ming Guo, Hugo J. Bellen, and Gregory Hannon for generously sharing fly lines. We thank Padmasini Chary and Rajeshwari Krishnamurthy (Bioklone Biotech Private Limited) for assistance with antibody generation. Fly lines obtained from the Vienna Drosophila Resource Center and the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. We also acknowledge the use of FlyBase (flybase.org ), a comprehensive database for Drosophila genetics and genomics, which was instrumental in the design and interpretation of this study. The MJ laboratory is supported by the Department of Atomic Energy, Government of India (Project Identification No. RTI 4007), the Department of Science and Technology, SERB/ANRF (CRG/2020/003275 and CRG/2023/005377), and the Department of Biotechnology (BT/PR32873/BRB/10/1850/2020). A.H. was supported by the Indo-German Grant by the Department of Biotechnology IC-12025( 11 )/2/2020-ICD-DBT. We thank Rajit Narayanan, Tarana Anand, Sunayana Sarkar and Yasir Hosein for valuable discussions and comments on the manuscript. Funder Information Declared Department of Atomic Energy, Government of India , RTI 4007 SERB/ANRF , CRG/2020/003275 , CRG/2023/005377 DBT-DFG Indo German Grant , IC-12025(11)/2/2020-ICD-DBT Department of Biotechnology, https://ror.org/03tjsyq23 , BT/PR32873/BRB/10/1850/2020 Bibliography 1. ↵ Madan S , Uttekar B , Chowdhary S , Rikhy R . Mitochondria lead the way: mitochondrial dynamics and function in cellular movements in development and disease . Front Cell Dev Biol . 2021 ; 9 : 781933 . 2. ↵ Sandoval H , Thiagarajan P , Dasgupta SK , Schumacher A , Prchal JT , Chen M , et al. Essential role for Nix in autophagic maturation of erythroid cells . Nature . 2008 Jul 10; 454 ( 7201 ): 232 – 5 . OpenUrl CrossRef PubMed Web of Science 3. Quiles JM , Gustafsson ÅB . The role of mitochondrial fission in cardiovascular health and disease . Nat Rev Cardiol . 2022 Nov ; 19 ( 11 ): 723 – 36 . OpenUrl CrossRef PubMed 4. ↵ Wang Z-H , Liu Y , Chaitankar V , Pirooznia M , Xu H . 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Share Developmental coordination of mitochondrial dynamics and membrane remodeling drives organelle morphogenesis H Aravind , Vivek Kumar , Manish Jaiswal bioRxiv 2025.06.09.658059; doi: https://doi.org/10.1101/2025.06.09.658059 Share This Article: Copy Citation Tools Developmental coordination of mitochondrial dynamics and membrane remodeling drives organelle morphogenesis H Aravind , Vivek Kumar , Manish Jaiswal bioRxiv 2025.06.09.658059; doi: https://doi.org/10.1101/2025.06.09.658059 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41913) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13372) Ecology (19889) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40365) Molecular Biology (17163) Neuroscience (88540) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15136) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9818) Zoology (2269)